G to A hypermutation is one of the characteristics of primate lentiviruses, as well as other retroviruses, during replication in vivo and in cell culture. The molecular mechanisms of this process, however, remain to be clarified. Recently, we have demonstrated that CEM15/APOBEC3G, an endogenous inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, is a cytidine deaminase and is able to induce G to A hypermutation in the newly-synthesized viral DNA. This effect can be counteracted by the HIV- 1 virion infectivity factor (Vif). We suggest that this viral DNA mutator may induce either "lethal hypermutation" or instability of the incoming nascent viral reverse transcripts, which could account for the Avifphenotype. Importantly, the accumulation of CEM15/APOBEC3G-mediated "non-lethal hypermutation" in the replicating viral genome could potently contribute to the genetic variation of primate lentiviral populations.To further investigate the molecular mechanism of hypermutationqnduced by CEM15/APOBEC3G, We will: (1). Further explore the mechanism of DNA deamination induced by CEM15. Especially, we will identify the substrate specificity of CEM15, analysis the components of the so called "editosome" by examining the possible interaction between CEM15 and viral proteins and examining the possible interaction between CEM15 and cellular proteins. The mechanism how CEM15 incorporates into HIV- 1 virions will be investigated. (2). Study the possible role of UDG in the generation of instability of nascent reverse transcripts. The hypothesis that the abasic site in the minus strand of viral DNA could be cleaved by apurinic/apyrimidinic(AP)-endonuclease will be examined. (3). Further determine the molecular mechanism of hypermutation occurring during viral passage in the cell culture. An RNA interference (RNAi) technique will be used to counteract CEM15 in the non-permissive cells. Investigate the possible effect of CEM15- mediated hypermutation upon the emergence of drug-resistant mutants. (4). Investigate the regulation of CEM15 gene expression. Especially, the promoter of CEM 15 will be identified and its activity will be examined. (5). Investigate whether other cytidine deaminases, such as AID, APOBEC1, APOBEC2, and APOBEC3A to 3F, etc, could inhibit the replication of HIV-1 and other retroviruses and induce hypermutation in the newly-synthesized viral DNA in the absence of v/f. We believe that these alternative but complementary approaches would enrich our knowledge regarding this anti-viral defense system. The research result from these projects will lead to find a novel strategy to combat HIV-1 replication.